Understanding the Application: NGS Panel Testing for Hereditary Cancer Syndromes and Cancer Targeted Therapy

Felicitas L. Lacbawan, MD, FCAP, FACMGMedical Director, Genetics Quest Diagnostics Nichols Institute, San Juan Capistrano, CA USA

February 9, 2016

Felicitas.L.Lacbawan@QuestDiagnostics.com

2

http://www.cancer.org/acs/groups/content/@editorial/documents/document/acspc-044552.pdf

http://www.nature.com/onc/journal/v23/n38/full/1207714a.html

5

About 5% to 10% of all cancers

Inheriting a gene mutation or pathogenic variant does not necessarily mean that a person will develop cancer, but it increases his/her risk

Most common hereditary cancers are: Breast cancer Ovarian cancer Colorectal cancer Prostate cancer

Understanding if cancer is due to an inherited pathogenic variant/mutation can help clarify future risks of developing cancer and help determine options for cancer screening and prevention, possibly therapy

Hereditary Cancers

http://seer.cancer.gov/

Can

cer R

isk

(%)

Cancer Type

Lifetime Cancer Risks for Common Cancers

Lifetime Risks: Breast Cancer

8

“Red Flags” for Inherited Susceptibility to Cancer

Cancer in 2 or more closely related relatives

Multiple generations affected

Early age at diagnosis

Multiple primary tumors

Bilateral or rare cancers

Constellation of tumors consistent with a specific cancer syndrome

Certain ethnic backgrounds (e.g. Ashkenazi Jewish ancestry)

9

Assessing Patient’s Family History

Ovarian, 52

Breast, 42 Breast, 45

Cost per Genome Decreasing Dramatically

11

Risk Management

• Diagnostic tests for complement traditional risk factors

Screening

• Applied to high-risk patients to identify disease early

Diagnosis

• Used for definitive diagnosis

Staging and Prognosis

• Assess severity

Therapy Selection

• Used to predict efficacy or safety response to specific treatments

Monitoring

• Monitoring for treatment efficacy

Diagnostic Applications of Sequencing

Test

Des

crip

tion

Testing Workflow for Cancer Gene Panel

DNA Extraction Library prep Target enrichment

SequencingReport Informatics

BloodFFPE cfDNA

13

Hereditary breast and/or ovarian cancer (HBOC) syndrome - most common high-risk breast cancer susceptibility syndrome

Mutations occur in 1:300 to 1:800 people 1:40 in Ashkenazi Jewish individuals

Cancer risks by age 70 y.o. for BRCA1 and BRCA2 mutation carriers without a personal history of cancer:

BRCA1 and BRCA2 Review

Mavaddat N et.al., Cancer Risks for BRCA1 and BRCA2 Mutation Carriers: Results from Prospective Analysis of EMBRACE. J Natl Cancer Inst. 105:812-822, 2013

Condition BRCA1 BRCA2Female Breast Cancer 55% to 65% 45% to 47% Ovarian Cancer 39% 11% to 17% Male Breast Cancer 1.2% 6.8%

14

Every year, more than 200,000 women in the U.S. will be diagnosed with breast cancer

Hereditary breast cancer accounts for about 5% to 10% of female breast cancer and 4% to 40% of male breast cancer

Genes with highest increased risk for breast cancer (highlighted):

Other Hereditary Breast Cancer Genes

Unexplained

50%

15%BRCA1BRCA2

14%Known SNPs

14%Other predicted SNPs

CHEK2ATMPALB2BRIP1RAD51CRAD51DBARD1

4%TP53PTENSTK11CDH1

3%

Fergus Couch et al 2014 Science 344, 1466

15

Breast Cancer Tumorigenesis

DNA repair – BRCA1/2, CHK2

Chromatin remodeling – BRCA1

Protein Ubiquitination - BRCA1

Cell cycle regulation – p53

Apoptosis – PTEN

Cell proliferation - PTEN

16

DNA Repair: BRCA1 and BRCA2

17

Gene Condition

BRCA1 and BRCA2 Hereditary breast and/or ovarian (HBOC) syndrome

TP53 Li-Fraumeni syndrome (LFS)

PTEN PTEN Hamartoma Tumor syndrome (PHTS), which includes Cowden syndrome (CS)

CDH1 Hereditary diffuse gastric cancer (HDGC)

STK11 Peutz-Jeghers syndrome (PJS)

PALB2 PALB2-associated breast cancer

Genes and Associated Syndromes

18

Lifetime Risk of Breast Cancer

TP53: breast cancer relative risk of 6.4x

PTEN : breast cancer risk of 85% by approximately age 70 y.o.

CDH1/ hereditary diffuse gastric cancer : lobular breast cancer risk of 39% to 52% by age 80 y.o.

STK11 : breast cancerrisk of 45% by age 70 y.o.

PALB2: breast cancerrisk of 35% by age 70 y.o.

Ruijs M et al 2010 J Med Genet 47: 421-248 Bubien V et al 2013 J Med Genet 50: 255-63Schrader KA et al 2008 Fam Cancer 7(1): 73-82Antoniou AC et al 2014 N Engl J Med 371(6): 497-506 Hearle N et al et al 2006 Clin Can Res 12;3209

19

Lifetime Risk of Other Key Cancers

Compared to the general population, TP53 relative risks of cancer:

Cowden syndrome and a mutation in PTEN risksof cancer by age approximately age 70:

Hereditary diffuse gastric cancer and a mutation in CDH1 gastric cancer risk of 40% to 67% in males and 63% to 83% in females

• Bone (107)• Connective tissue (61)• Brain (35)

• Thyroid (35%)• Endometrial (28%)• Renal (34%)

Peutz-Jeghers syndrome –gastrointestinalcancer risk of 57% (includes pancreatic);pancreatic cancer risk of 11% by age 70.

• Pancreas (7.3)• Colon (2.8)• Liver (1.8)

• Colorectal (9%)• Melanoma (6%)

McBride KA et al 2014 Nature Reviews Clin Oncology 11, 260-271Bubien V et al 2013 J Med Genet 50: 255-63Caldas C et al. 1999 J Med Genet 36(12):873-80. Kaurah P et al. 2007 JAMA 6;297(21): 2360-72Hearle N et al et al 2006 Clin Can Res 12;3209

20

Genetic Testing Criteria: HBOC

National Comprehensive Cancer Network. NCCN clinical practice guidelines in oncology. Genetic/familial high-risk assessment: breast and ovarian. V2.2014.

21

BRCA1 or BRCA2(Hereditary Breast and/or Ovarian Cancer Syndrome)

Individual from a family with a known deleterious BRCA1 or BRCA2 mutation

Personal history of breast cancer diagnosed at age ≤45 y**

Personal history of breast cancer with additional criteria**

Personal history of epithelial ovarian cancer

Personal history of male breast cancer Personal history of pancreatic cancer or

prostate cancer with additional criteria Additional criteria for patients without a

personal history of cancer, with a family history of HBOC-related cancers

Women Clinical breast exam: every 6 to 12 months,

starting at age 25 y Age 25 to 29 y: annual breast MRI Age >30 to 75 y: annual breast MRI and

mammogram Discuss risk reducing mastectomy Recommend risk-reducing salpingo-

oophorectomy Consider chemoprevention options

Men Clinical breast exam: every 6 to 12 months,

starting at age 35 y Consider baseline mammogram at 40 y Prostate cancer screening starting at age 40 y

Additional recommendations

NCCN Genetic Testing Criteria*

NCCN Management Guidelines*

* NCCN clinical practice guidelines in oncology. Genetic/familial high-risk assessment: breast and ovarian. V2.2014.

** Includes invasive and ductal carcinoma in situ breast cancers.

22

Genetic Testing Criteria and Management Guidelines

Gene Genetic Testing Criteria Management GuidelinesBRCA1 and BRCA2 NCCN: Genetic/Familial High-Risk Assessment:

Breast and OvarianTP53PTEN

CDH1 International Gastric Cancer Linkage Consortium consensus guidelines

STK11 None NCCN: Genetic/Familial High-Risk Assessment: Colorectal

PALB2 None

ACS recommends screeningwith MRI for women with at least 20% to 25% lifetime risk of breast cancer

Shiovitz & Korde LA, 2015 Ann Oncol

23

BRCA1 and BRCA2 may explain 15% to 20% of hereditary breast cancer cases

TP53, PTEN, CDH1, STK11, and PALB2: breast cancer susceptibility genes together explain an additional 3% to 4.5% of hereditary breast cancers

Focused risk-assessment options exist for guideline-supported and emerging genes Guideline supported: BRCA1, BRCA2, TP53, PTEN, CDH1, STK11

Emerging: PALB2 (Antoniou et al 2014 N Engl J Med 371(6): 497-506)

Breast Cancer Expanded Menu

24

When to Consider Multi-Gene Testing

Advantages: decreased cost and improved efficiency of cancer genetic testing by decreasing the time involved, number of patient visits, and number of tests sent

Primary disadvantage: increased complexity of results

Genetic counselors or knowledgeable medical professionals should carefully discuss the pros and cons with patients

Additional recommendations

The decision to use multi-gene testing for patient care should be no different than the rationale for testing a single gene known to be associated with the development of a specific type of cancer

Multi-gene testing may be more cost-effective and time-effective in certain cases than sequentially testing more than 2 to 3 single genes associated with a phenotype

BRCA1/BRCA2, TP53, and PTEN: Consider multi-gene testing, if appropriate

Additional recommendations

Society of Gynecologic Oncology (SGO) Clinical Practice Statement: Next Generation Cancer Gene Panels Versus Gene by Gene Testing2 (March 2014)

National Comprehensive Cancer Network (NCCN) Guidelines V2.2014 Genetic/Familial High-Risk Assessment: Breast and Ovarian, GENE-1 (September 2014)

25

Hereditary Breast Cancer Panel Test Options: Part 1

Comprehensive Includes sequencing and large rearrangement analysis of all coding exons in BRCA1 and BRCA2

AshkenaziJewish Screen

Includes detection of the 3 HBOC foundermutations (187delAG, 5385insC, 6174delT)

Rearrangements BRCA1 and BRCA2 complete rearrangement testing not performed

Single Site Specific mutation testing for a knownfamilial mutation

Ashkenazi Jewish Screen with Reflex Comprehensive

Ashkenazi Jewish screen test that reflexes to the comprehensive test when Ashkenazi test is negative

26

Test Name Clinical ApplicationBRCA Expanded PanelIncludes point mutations, deletions, and duplications in the BRCA1, BRCA2, TP53, PTEN, CDH1, STK11 and PALB2 genes

• Assess hereditary breast cancer risk when there is no known familial mutation and the patient has a personal or family history consistent with more than 1 condition related to hereditary breast cancer

• Simultaneous analyses of relevant genes

BRCA w/ Reflex to Breast Plus PanelIncludes test code 91863; test code 92586 added with additional charge and CPT code, if no BRCA1 or BRCA2 mutations detected

• Assess hereditary breast cancer risk when no known familial mutation and the patient has a personal or family history consistent with more than 1 condition related to hereditary breast cancer

• Two-step analysis begins with testing for mutations in BRCA1 and BRCA2, the most common causes of hereditary breast cancer; 5 additional genes analyzed if pathogenic or likely pathogenic mutations not detected in the first step

BRCA Expanded Panel w/o BRCA 1/2Includes point mutations, deletions, and duplications in the TP53, PTEN, CDH1, STK11, PALB2 genes

• Second-tier test to assess hereditary breast cancer risk in people negative for BRCA1 and BRCA2 point mutations, deletions, and duplications

Hereditary Breast Cancer Panel Test Options: Part 2

Cancer Predisposition Panel: 34 Genes

28

Clinical Actionability: HBOC Risk Assessment

Desmond et al., 2015 JAMA Oncol

Desmond, 2015 LAB A LAB B LAB C

# Genes 29 25 34 34BRCA1 BRCA1 BRCA1BRCA2 BRCA2 BRCA2

High risk BR and OV CA

TP53 TP53 TP53 TP53PTEN PTEN PTEN PTENSTK11 STK11 STK11 STK11CDH1 CDH1 CDH1 CDH1

Low-mod risk BR and

OV CA

BARD1 BARD1 BARD1 BARD1CHEK2 CHEK2 CHEK2 CHEK2PALB2 PALB2 PALB2 PALB2 (FANCN) ATM ATM ATM ATMBRIP1 BRIP1 BRIP1 BRIP1RAD51C RAD51C RAD51C RAD51CRAD51D RAD51D RAD51DNBN NBN NBN NBN (NBS1),

Lynch syndrome

MLH1 MLH1 MLH1 MLH1MSH2 MSH2 MSH2 MSH2MSH6 MSH6 MSH6 MSH6PMS2 PMS2 PMS2 PMS2EPCAM EPCAM EPCAM EPCAM

Other Familial

APC APC APC APCBMPR1A BMPR1A BMPR1A BMPR1ASMAD4 SMAD4 SMAD4 SMAD4CDK4 CDK4 CDK4 CDK4CDKN2A CDKN2A (p16,

p14)CDKN2A CDKN2A (p16, p14)

MUTYH Biallelic MUTYH MUTYH MUTYH (MYH)

MET METMEN1 MEN1 MEN1RET RET RETPTCH1 PTCH1VHL VHL VHL

FANCCNF1 NF1

RAD50SDHBSDHCSDHDPOLD1POLE

PALLD

29

Assay validation Dictated by assay design, panel content, gene structure (i.e. CHEK2 & PMS2

pseudogenes)

Accuracy, sensitivity, specificity, limits of detection

Efforts in reducing sequencing errors, increase capture

Limitations of platform used – mosaicism, low copy number variants (somatic)

Type of mutation and rearrangements – triplet repeats, CNVs, large rearrangement (inversion/translocation)

Sequencing performance/Quality metrics

Alignment software for accurate allele identification Detection ability - ≥10bp deletions and insertions

Analytical Performance Review

30

Variant classification using the American College of Medical Genetics (ACMG) 5-tier classification system

Multiple data sources ClinVar archive Breast Cancer Information Core (BIC) Universal Mutation Database (UMD) Leiden Open Variation Database (LOVD) Prediction tools such as PolyPhen-2, SIFT, Align GVGD, and MutationTaster

Final interpretation performed by board-certified directors

Multiple reviews for VUS and pathogenic cases

Co-segregation family study program to help with VUS reclassification

Reclassified variants will be communicated to the ordering provider when a patient result is amended

Variant Annotation & Classification

31

Breast Cancer: Report

Turnaround time (TAT)

• Up to 14 days (upon receipt of complete requisition)• Reflex test option up to 21 days (upon receipt of complete requisition)

Content

• Interpretation Summary• Color-Coded 5-Tier Classification

o Known Pathogenic (RED)o Likely Pathogenic (RED)

• VUS (YELLOW)• Likely Benign Polymorphism (GREEN)• No Mutation Detected (GREEN)• Comprehensive Interpretation• ACMG Guidelines

32

Solid Tumors

Multiple genes and pathways altered

Tumor suppressor genes and oncogenes

Targeted therapies available

Clinical annotation and clinical utility must be established

33 Hanahan & Weinberg Cell 144, 643 (2011).

Clinical View of Cancer

34

Cancer Pathways and Targeted Treatments

KIT/

GIST

CRC Multiple

RCC/GIST

RCC/ HCC

Breast Ca

Lung Ca

RCC

35

Solid Tumor Gene Mutations Related to Therapy

ERBB4

BladderFGFR3

Cervical

Endometrial

FOXL2

Thyroid

HRAS

HRAS

IDH1IDH2

FGFR2

PIK3R1

GNASPancreas

FGFR4

NOTCH1STK11

TP53

Multiple

PTCH1FBXW7

RET

36

More Common Solid Tumor Genes

37

Solid Tumor by NGS

Targeted actionable genes

34-gene panel with broad mutation representation

Panel applicable to all solid tumor types

Annotation directed at FDA-approved drugs in selected tumor types and clinical trial availability

FFPE tissue, small biopsies, FNAs

38

Lung Cancer

Treatment Options Based onMolecular Profile

EGFR exon 18, 19 or 21 mutation

Tarceva® (erlotinib)

EGFR T790MKRAS codon 12, 13,

61, etc.

39

Current Tumor Panels

Lung Panel

EGFR

BRAF

ALK

NRAS

HRAS

KRAS

PIK3CABRAF

KIT

BRAF

Colorectal Panel

34 Gene NGS Panel

1 Assay (34 genes; >230 amplicons)

(ROS1/RET/MET FISH)

(+ RAS, HER2 mutation)

Melanoma Panel

40

Level 1 Associations Between Genes and FDA-Approved Therapies

Gene Mutation Drugs Tumor Type AssociationBRAF Vemurafenib Melanoma SensitiveEGFR Cetuximab Head and Neck Sensitive

EGFRGefitinib, Erlotinib, Afatinib, Cetuximab Lung

Sensitive or resistant depending on mutation

HRAS VemurafenibCutaneous Squamous Cell Carcinoma Resistant

KIT

Imatinib, Sunitinib, Regorafenib, Sorafenib

Gastrointestinal Stromal Tumor (GIST) Sensitive

RETVandetanib, Cabozantinib,  Thyroid Sensitive

SMO Vismodegib Basal Cell Carcinoma (skin) Sensitive

KRASCetuximab, Panitumumab Colorectal Resistant

NRASCetuximab, Panitumumab Colorectal Resistant

41

Gene Targeting

|

Gene Treatment MarkerAKT1 PI3K/mTOR/AKTALK ALK inhibitors, incl. crizotinib, Xalkori Gene & Resistance

BRAF RAF inhibitors, MEK inhibitors, PI3K inhibitors GeneCTNNB1 mTor inhibitors GeneDDR2 Some TYR-Kinase inhibitors, Nilotinib Other, GeneEGFR EGFR inhibitors, EGFR antibodies Gene

ERBB2 anti HER2,ERBB2 inhibitors, ERBB2 antibodies GeneERBB4 lapatinib Gene

ESR1 Associated with resistance to anti-estrogen ResistanceFGFR2 FGFR inhibitors, FGFR antibodies GeneFGFR3 FGFR inhibitors, FGFR antibodies Gene

HRAS RAF inhibitors, MEK inhibitors, PI3K inhibitors ResistanceIDH1 IDH1 inhibitor Gene & OtherKIT imatinib/sunitinib Gene

KRAS RAF inhibitors, MEK inhibitors, PI3K inhibitors Resistance

MAP2K1 RAF inhibitors, MEK inhibitors, PI3K inhibitors (eg. Mekinist) GeneMET MET inhibitors, MET antibodies Gene

NRAS RAF inhibitors, MEK inhibitors, PI3K inhibitors ResistancePDGFRA Kinase Inhibitors, Antibodies Gene

PIK3CA PI3K inhibitors, AKT inhibitors,mTor inhibitors Gene & PathwayPIK3R1 PI3K inhibitors, AKT inhibitors PathwayPTEN PI3K inhibitors PathwaySMAD4 MEK-ERK, p38-MAPK PathwaySMO observed vismodegib resistance, GeneVHL VEGF inhibitors Pathway

42

Clinical Applications of NGS Multigene Cancer Panel

Primarily for cancer patients with few or no standard treatment optionsremaining. Assist oncologist decision on potentially effective drug or clinicaltrial that would not have been previously considered.

• All solid tumor types may be tested

• Metastatic or locally advanced disease at presentation

• When no actionable mutations in guideline-recommended testing

• Small specimens without sufficient material for all guideline-recommended studies to be completed

• Recurrent or metastatic disease that has progressed through all standard of care options

• Tumors of unknown primary origin

• Rare tumor types where no or few standard of care options exist

43

Actionability: Results That Guide Decision Making

An evolving concept, varying with patient, clinician, guideline committee,

and payer

• Is contextual for stage of disease (primary vs metastatic) and tumor type

• Guidelines and FDA-approved drug labels formally define accepted criteria

• Inclusion in a clinical trial may be considered actionable

• Anticipation of additional genes/mutants that may be actionable in near

future is necessary

• Actionability is not binary but is best thought of as a continuum of evidence

44

Multigene NGS Cancer Panel

Solid Tumor Mutation Panel

AKT1 FGFR2 IDH2 PIK3R1

ALK FGFR3 KIT PTCH1

BRAF FGFR4 KRAS PTEN

CTNNB1 FOXL2 MAP2K1 RET

DDR2 GNA11 MET SMO

EGFR GNAQ NOTCH1 STK11

ERBB2 GNAS NRAS TP53

ERBB4 HRAS PDGFRA

FBXW7 IDH1 PIK3CA

Answers to guide treatment of most solid tumors, including approved or investigational targeted therapies

Actionable genes: approved targeted therapies, NCCN guidelines, clinical trials, prognostic indications

34 genes; >230 amplicons

Advantages

– Multiple genes interrogated simultaneously

– Enhanced sensitivity over Sanger sequencing

– Multiplexing patient samples reduces cost of sequencing

– Targeted sequencing with modifiable content and verification

45

Assay Characteristics

Released March 24

Formalin-fixed and paraffin-embedded

sections

2-5 X 5 um X 1 cm2 (5-20 ng)

Sensitivity 5%*

Verification (Sanger, PCR, MiSeq)

Alternate Specimen Types (FNA, cells, etc.)

* 5% against wild-type background.

46

Specimen Flow

Sectioning, Staining, Pathologist Review and Macrodissection

DNA Extraction and Quantitation

Validated sample types (FFPE, FNA, cells)

47

Mutation Distribution in Common Cancer Types

Quest Diagnostics internal data on file.

n=30 each

48

Annotation Goal Is to Simplify Complex Information

ComplexBiology

SimpleDirection

• 34 Genes• 100s of mutations• 1000s of scientific

papers• FDA-approved and

investigational cancer drugs

• Clinical trials• Guidelines

49

Multigene Tissue (Somatic) Report

Mutations identified

Clinical relevance

National and international guidelines

Treatment options: tumor type-specific and additional tumor types

Clinical trials

Level of evidence (e.g. publications)

50

Primary tumors are heterogeneous

Metastases differ from primaries

Tumors can “evolve” and become resistant

Individual may need multiple tests

Reimbursement

It’s Not so Simple….Challenges

51

Other Approaches

Larger Panels 300 – 400 genes

Whole Genome/Whole Exome Sequencing With or without comparison to germline

Liquid Biopsy (free floating tumor DNA) Hot spot (mutation) analysis

Sequencing

Can be used for primary diagnosis, drug selection, or monitor recurrence

52

During recent years, access to genetic testing for cancer predisposition and solid tumor genetic profile has broadened

Important technical advances have made it possible to perform multi-gene sequencing assays; test performance may vary according to performing laboratory test validation design and platform used

Existing mutation databases and a newly created public/private organization are expected to enhance the clinical utility of genetic results

Overall, the field of genetic testing for predisposition to cancer is becoming fundamentally important and proving clinical validity and utility

Solid tumor genetic profile influence drug selection, enabling targeted therapy

Summary

53

Thank You for Your Attention!